As early as 1953, ZoBell suggested that sulfur be removed from petroleum hydrocarbons by reduction and removal of hydrogen-reducible compounds by use of microbiological catalysts such as hydrogenases, including hydrogenases produced by sulfate-reducing microorganisms such as Desulfovibrio desulfuricans and Sporovibrio (U.S. Pat. No. 2,641,564). In one embodiment, he suggested that hydrogen for the reduction step be produced in situ by the action of a Clostridium, Aerobacter or other microorganism on carbohydrates (ibid., column 4, line 75 to column 6, line 25). Later researchers confirmed that sulfate-reducing bacteria can remove sulfur from petroleum using electrochemically supplied electrons (e.g., Desulfovibrio desulfuricans M6 described in Kim, et al., 1990, Biotechnol. Lett. 12: 757-760).
Though the validity of using an unsubstituted, low molecular weight model compound to select candidate microorganisms for desulfurization testing remains uncertain, because of its abundance in coal and oil and its refractory nature in chemical and physical desulfurizations, dibenzothiphene (herein abbreviated DBT) has become useful as a model for studying petroleum biodesulfurization (Fought, cited above, pp. 387-388). DBT is metabolized in bacteria by different metabolic pathways. In one pathway, a portion of the carbon DBT is metabolized without carbon-sulfur bond cleavage (id.). This pathway is undesirable from the standpoint of fossil fuel cleaning, since a portion of the organic structure of the molecule is destroyed and carbon-sulfur bond cleavage does not occur.
Another pathway of DBT metabolism is also carbon destructive, but carbon-sulfur bond cleavage occurs, releasing sulfite and sulfate. A Brevibacterium species denoted DO, for example, was reported to metabolize DBT as a sole source of carbon, energy, and sulfur, releasing sulfite (van Afferden, et al., 1990, Arch. Microbiol. 153: 324-328). Arthrobacter K3b metabolized DBT sulfone by a similar pathway, but was incapable of removing oxidized organic sulfur from Illinois No. 6 coal (Dahlberg, M. D., et al., 1993, Fuel 72: 1645-1650). Sulfolobus acidocaldarius reportedly oxidized DBT, forming sulfate (Kargi, F., and Robinson, J. M., 1986, Fuel 65: 397-399), but organic products of this metabolism were not determined, and it was unclear if the metabolism was carbon destructive. The organism reportedly removed 44% of organic sulfur from coal, although results with uninoculated controls were not reported.
Organisms that remove sulfur from DBT without destruction of the carbon ring structure appear to be most promising for possible application to fossil fuel desulfurization. Isbister and Kobylinski described a strain of Pseudomonas that produced sulfate and dihydroxybiphenyl from DBT (deposited in the A.T.C.C. as accession number 39381, U.S. Pat. No. 4,562,156 to Isbister, J. D., and Doyle). Isbister subsequently described an Acinetobacter strain CB2 (A.T.C.C. # 53515) that oxidized diphenyl sulfide and acted on organic sulfur in coal, but did not appear to remove thiophenic sulfur (U.S. Pat. No. 4,808,535).
Isolation after a selective mutation process led to the development of mutant Rhodococcus rhodochrous strain IGTS8 (U.S. Pat. No. 5,104,801 to Kilbane), an organism that metabolizes DBT via a pathway not destructive of the carbon rings of the molecule, and generated significant interest in the potential use of microorganisms for fossil fuel desulfurization. This organism has been deposited in the A.T.C.C. (accession no. 53968) and is the most widely studied bacterium considered for possible application to fossil fuel desulfurization. The organism metabolizes DBT as a sole source of sulfur, forming either 2-hydroxybiphenyl or 2,2'-dihydroxybiphenyl as a product (ibid.; Gallagher, J. R., et al., 1993, FEMS Microbiol. Lett. 107: 31-36; and Olson, G. J., 1993, Fuel Processing Technol. 40: 103-114), thus leaving the hydrocarbon portion of the molecule intact. Carbon-sulfur bond cleavage in DBT occurs, but sulfur does not accumulate in solution; it is apparently used immediately by the cells for biosynthesis (Gallagher, cited above).
The metabolic pathway of DBT degradation in R. rhodochrous IGTS8 under growing conditions proceeds via oxidation of the sulfur to the sulfoxide, sulfone, sulfonic acid, and the desulfurized product, 2,2'-dihydroxybiphenyl. Under resting conditions, the cells produce sulfoxide, 2'-hydroxybiphenyl-2-sulfinate and 2-hydroxybiphenyl (ibid.). The organism has been tested for its ability to desulfurize coal (Kilbane and Bielaga, cited above) and solubilized coal (Kilbane, J. J., and Jackowski, K., 1991, Biotechnol. Bioengin. 40: 1107-1114). However, since the organism uses sulfur in an assimilatory fashion, experimental systems require large amounts of cells to be applied to small amounts of coal. This limits the potential usefulness of a whole cell approach to coal biodesulfurization with this organism.
Moreover, it has generally been impossible to fully evaluate the validity of reports about organic sulfur removal from coal because insufficient data arepresented in support of the findings. The experimental deficiencies include reliance on indirect "organic sulfur" measurements, lack of appropriate experimental controls, and absence of data on coal recovery and associated elemental analyses (Olson, cited above, p. 104). Measurement of the "organic sulfur" content of coal is especially subject to error because the presence of elemental sulfur, metal sulfides, jarosites, and pyrites in some coals all lead to errors in indirect organic sulfur measurements. This is particularly true where significant biomass is associated with coal samples used because, since biomass has a much lower sulfur content than many coals, apparent desulfurization may occur due to dilution.
Nonetheless, R. rhodochrous or enzyme and/or membrane extracts of R. rhodochrous cultures (U.S. Pat. Nos. 5,132,219 and 5,344,778 to Kilbane) have been suggested in processes for the cleavage of organic C-S bonds in oil or coal (U.S. Pat. No. 5,358,869 to Kilbane), including continuous processes involving oxygenation and regeneration steps (U.S. Pat. No. 5,472,875 to Monticello), in combination with hydrodesulfurization for the "deep desulfurization" of liquid fossil fuels (U.S. Pat. Nos. 5,232,854 and 5,387,523 to Monticello), and in a process for the desulfurization and desalting of fossil fuels (U.S. Pat. No. 5,356,813 to Monticello).
Following publication of Kilbane's work on R. rhodochrous, other organisms have been described which appear to have similar metabolic pathways in the desulfurization of dibenzothiophene. These include a Bacillus sphaericus strain (A.T.C.C. Accession No. 53969, U.S. Pat. No. 5,002,888 to Kilbane), a strain identified as Corynebacterium sp. strain SY1 (Omori, T., et al., 1992, Appl. Environ. Microbiol. 58: 911-915), and a bacterium tentatively identified as R. erythropolis D-1 (Izumi, Y., et al., 1994, Appl. Environ. Microbiol. 60:223-226). These reportedly utilize DBT as a source of sulfur and produce 2-hydroxybiphenyl, releasing small amounts of sulfate. B. sphaericus A.T.C.C. 53969 requires a nutritional helper bacterium in order to metabolize DBT. Other strains of R. erythropolis have been described which convert DBT to monohydroxybiphenyl (Wang, P., and Krawiec, S., 1994, Arch. Microbiool. 161: 266-271); the presence of sulfate in the growth media repressed expression of desufurization activity, but sulfate added to suspensions of cells grown in DBT did not inhibit desulfurization activity. Gordona strain CYKS1 converts DBT to 2 hydroxybiphenyl (Rhee, S.-K., et al, 1998, Appl. Environ. Microbiol., 64: 2327-2331) Like Rhodococcus strain IGTS8, DBT metabolism was repressed by sulfate. The organism reportedly removed sulfur from diesel oils, although results from uninoculated controls were not given.
It would be desirable to have other, improved biocatalysts for the desulfurization of fossil fuels, especially agents that do not metabolize the fuel itself as a carbon and/or energy source. It would also be desirable to have fuel biodesulfurization agents isolated from natural sources that are economical and easy to cultivate.